biotechnology -- chap. 16. the use of biological systems for the production of materials (most work...
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Biotechnology -- Chap. 16.The use of biological systems for the
production of materials (most work is in the field of Genetic Engineering)
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• process of altering biological systems by the purposeful manipulation of DNA – introduce specific foreign pieces of DNA into
host cells (often bacteria or viruses) which is then replicated by the host cells
– as the cell divides, a clone (exact copy) is produced
– cells containing the new DNA can be grown in any quantity and therefore, so is the protein transcribed by that DNA.
Genetic engineering
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• The hard part is to “convince” the host cell to accept the foreign DNA– this is done by attaching the foreign DNA to a
carrier DNA molecule called a vector. – vectors are often bacteria’s plasmid (once
together they are now recombinant DNA) • Plasmids are molecules of DNA that are found in
bacteria separate from the bacterial chromosome. They: • are small, carrying one or a few genes• are circular • self-replicating
• This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.
The Process
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• enzymes that cut the phosphate-backbones of DNA at specific base sequences (normally 4-6 bases) called restriction sites.
• exist naturally in bacteria in order to cut apart invading viral DNA
• cut parts of DNA are called restriction fragments
Restriction enzymes (also called restriction endonucleases)
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• naming: ex) EcoRI– 1st letter is for genus - (Escherichia)– 2nd letter/s is for species - (coli)– 3rd letter is for the strain of the organism (R)– Roman numerals at the end typically
indicates the order of discovery (I)
Recombinant DNA video
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Splicing and Cloning DNA Molecules (Fig. 16.2 in text)
• a restriction fragment of DNA can be duplicated by inserting it in a vector DNA molecule
• most common vectors are plasmids in bacteria and DNA viruses
• first, the vector and foreign DNA are cleaved by the same restriction enzyme in order for the sticky ends of both to match and then mixed together
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• The enzyme ligase is added to join the vector and foreign DNA together making the foreign DNA an integral part of the plasmid
• This new plasmid is then taken up by other bacteria cells by a process called transformation
• Identification: Often the foreign DNA that was just inserted has other segments of DNA that identify it, such as resistance to antibiotics and the ability to produce color.
Steps in Cloning a Gene
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DNA Sequencing
• the determination of the precise sequence of nucleotides in a sample of DNA.
• dideoxynucleotides are synthetic nucleotides (4 types, ddATP, ddGTP, ddCTP, ddTTP) that lack the -OH at the 3′ carbon atom and are each labeled with a "tag" that fluoresces a different color
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• chain elongation proceeds normally until by chance, DNA polymerase inserts a dideoxynucleotide instead of the normal deoxynucleotide
• Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence
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